The explosion of recent knowledge in basic genetics has spawned numerous clinical follow-up studies that have confirmed an unequivocal association between the presence of specific prevalent genetic alterations and susceptibility to some very common human diseases. In addition, the Human Genome Project's sequencing efforts will contribute yet more candidate disease genes that will require both research-based genetic association studies (to confirm suspected disease links) and, if positive, the translation of these disease-genotype associations to routine diagnostic clinical practice. Given this expanding repertoire of confirmed and reputed disease genes (many for common diseases), the demand for rapid, sensitive, specific, inexpensive assays for their clinical- and/or research-based detection is growing quickly.
As a consequence, clinical genetic testing laboratories, once accustomed to manual, low-volume, high-labor tests on patients with rare, untreatable classic “genetic” diseases, will soon need to develop better high-throughput and semi-automated methods. In the fast-approaching molecular medicine era, these new genotyping methods will be utilized not only for diagnosing symptomatic patients but perhaps, more importantly, for presymptomatically identifying individuals at risk for common, treatable diseases for whom effective preventative interventions may be available.
Oligonucleotide hybridization is a method commonly used in the field of molecular biology for the treatment and diagnosis of disease, as well as the identification, quantitation, and isolation of nucleic acids. Accordingly, it is important to identify methods to increase the specificity and affinity of oligonucleotides for their targets. In this way, diagnostics which provide efficient and precise answers can be made. Various methods for increasing the specificity of oligonucleotides are known in the art, including increasing the length, choosing oligonucleotides that are not likely to cross-hybridize or bind non-specifically and designing oligonucleotides that have a high annealing temperature. (See e.g., Bergstrom et al., J. Am. Chem. Soc. 117:1201-1209, 1995; Nicols et al., Nature 369:4920493, 1994; Loakes, Nucl. Acids Res. 22:4039-4043, 1994; Brown, Nucl. Acids Res. 20:5149-5152, 1992).
Recently, investigators have determined that modified oligonucleotides containing universal bases provide some benefit over conventional oligonucleotide chemistries. (See Guo et al., U.S. Pat. No. 5,780,233, filed Jun. 6, 1996). Although Guo et al., observed some improvement in being able to discriminate a variant nucleotide in a target nucleic acid by incorporating solitary universal bases (artificial mismatches) sprinkled throughout a probe oligonucleotide, particular spacing and composition requirements were necessary. For example, Guo et al. found that the universal base should be carefully spaced from the variant nucleotide (i.e., 3 or 4 nucleotides away) and that the oligonucleotide probes should not contain a total composition of universal bases of greater than 15%.
Van Ness et al. (U.S. Pat. No. 6,361,940, filed Apr. 1, 1998) also found that the incorporation of universal bases (specificity spacers) could increase the specificity of a probe oligonucleotide for a target nucleic acid. As above, however, Van Ness et al. determined that the universal bases should be spaced a considerable distance from each other (4-14 nucleotides). Thus, despite the advances made by the investigators above and others in the field, there still remains a need for better oligonucleotide chemistries, which allow for the development of more efficient diagnostics and therapeutics.